The primary goal of this proposal is to explore the hypothesis that Notch4 contributes to the integrity of the mature podocyte. This will be accomplished through an analysis of in vitro and in vivo models of antibody-mediated podocyte injury and Notch4-deficient (N4-/-) mice. We found that Notch4 is present in the podocytes of mature rats and mice and that the podocytes of N4-/- mice are focally effaced with increased expression, clustering and dislocation of nephrin. Moreover, Notch4 expression is abundant and total nephrin is reduced coincident with the onset of proteinuria in rats with passive Heymann nephritis, which suggests that Notch4 might regulate nephrin gene transcription. Thus, we will examine the role of Notch4 in the mature podocyte according to four specific Aims. In the first specific Aim we will use an in vitro model of antibody- and complement-mediated cell injury to determine if Notch4 is activated by gammasecretase and translocates to the nucleus to bind and activate its nuclear target, CSL In the second specific Aim we will examine N4-/- mice overtime to determine if Notch4 deficiency leads to proteinuria and alterations in podocyte morphology and podocyte-associated proteins. We will also establish if Notch4 regulates nephrin expression in vitro using a nephrin promoter reporter system in 293 cells, and in vivo in N4-/- mice. The third specific Aim is to determine if Notch4 influences the susceptibility to and/or recovery from immune glomerular injury. N4+/+ and N-/- littermates will be studied using various models of antibody-mediated podocyte injury. Their susceptibility to injury and time to recovery will be monitored, and the effect of gamma-secretase inhibition will be examined. The fourth specific Aim will utilize a comparative transcriptome analysis of glomerular RNA derived from young male N4+/+ and N-/- littermates to identify genes that Notch4 regulates in mature glomeruli. Differentially expressed genes will be examined for appropriate location and expression in glomeruli and regulation by Notch4. The results of these studies may disclose mechanisms underlying premature podocyte degeneration and glomerular sclerosis and the variable clinical and pathological response of patients with antibody-mediated podocyte injury.